Drug-induced hepatotoxicity is the most common cause of acute liver injury, a potentially fatal condition for which current therapies are limited o supportive care and liver transplantation. It is also the most common reason that potentially life-saving drugs are abandoned during development and never reach patients. Heprotech Inc. was formed to develop liver-safe pharmaceuticals and treatments for drug-induced liver injury. We recently discovered a novel role for gap junction-dependent communication in the propagation of drug-induced liver injury and have identified a tool compound that interferes with this pathway. Heprotech now seeks funding to support a drug discovery program that will use high throughput screening of small molecule libraries to develop a portfolio of therapeutic compounds that will serve as first-in-class hepatoprotectants. The overall goal of this project is to developa high throughput screening assay that will identify a novel class of drugs that will be developed to treat and prevent drug-induced liver injury. This grant focuses on discovery of inhibitors of connexin 32 (Cx32), a liver-specific gap junction protein that we recently demonstrated to be essential for propagating drug-induced liver injury. The project's specific aims are: 1) To develop a Cx32-containing liposome reporter system for measuring gap junction permeability, 2) To establish a reproducible high throughput screening assay using the Cx32 liposome reporter system, and 3) To screen for inhibitors of Cx32 hemichannels and evaluate their ability to limit propagation of cellular injury in a live cell-based secondary screen. The anticipated outcome of this effort is a collection of promising potent Cx32 inhibitors that can be further developed through rigorous preclinical studies during phase II of this SBIR program.